Methods of treating drug resistant and other tumors by administering 6,7-dialkoxy quinazoline derivatives

ABSTRACT

Methods employing and uses of a compound of formula (I) in inhibiting the growth of a tumor cell in a subject in need thereof. Methods employing and uses of a compound of formula (I) in treating pancreatic cancer in a subject in need of treatment for pancreatic cancer. Methods employing and uses of a compound of formula (I) in treating HER-2 positive breast cancer in a subject in need of treatment for HER-2 positive breast cancer. Methods employing and uses of a compound of formula (I) in treating drug resistant non-small cell lung cancer in a subject in need of treatment for drug resistant non-small cell lung cancer. Each of these methods can include administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. 
     Method of manufacturing a medicament including a compound of formula (I) for treating a subject suffering from growth of a tumor cell; for treating a subject suffering from pancreatic cancer; for treating a subject suffering from HER-2 positive breast cancer; or for treating a subject suffering from drug resistant non-small cell lung cancer.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of Ser. No. 61/225,419, filed Jul. 14, 2009 in the United States and which application is incorporated herein by reference.

FIELD OF THE INVENTION

Methods employing and uses of a compound of formula (I) in inhibiting the growth of a tumor cell in a subject in need thereof. Methods employing and uses of a compound of formula (I) in treating pancreatic cancer in a subject in need of treatment for pancreatic cancer. Methods employing and uses of a compound of formula (I) in treating HER-2 positive breast cancer in a subject in need of treatment for HER-2 positive breast cancer. Methods employing and uses of a compound of formula (I) in treating drug resistant non-small cell lung cancer in a subject in need of treatment for drug resistant non-small cell lung cancer. Each of these methods can include administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

Method of manufacturing a medicament including a compound of formula (I) for treating a subject suffering from growth of a tumor cell; for treating a subject suffering from pancreatic cancer; for treating a subject suffering from HER-2 positive breast cancer; or for treating a subject suffering from drug resistant non-small cell lung cancer.

BACKGROUND OF THE INVENTION

Most of the treatment regimes of the past for cell proliferation diseases such as psoriasis and cancer utilize compounds which inhibit DNA synthesis. Such compounds are toxic to cells and their beneficial effects can be derived when they show selectivity to tumor cells. In recent years it has been discovered that a cell may become cancerous by virtue of the transformation of a portion of its DNA into an oncogene, i.e., a gene which, on activation, leads to the formation of malignant tumour cells (Bradshas, Mutagenesis, 1986, 1:91). Several oncogenes encode tyrosine kinase enzymes and certain growth factor receptors are also tyrosine kinase enzymes (Larsen et al., Ann. Reports in Med. Chem. 1989, Chapt. 13).

Receptor tyrosine kinases are important in the transmission of biochemical signals which initiate cell replication. They possess an extra cellular binding domain for growth factors such as an epidermal growth factor and an intracellular portion which functions as a kinase to phosphorylate tyrosine amino acids in proteins and hence to influence cell proliferation. Members of the ErbB family of receptor tyrosine kinases are mediators of cell growth, differentiation and survival that have been implicated in cancer. The receptors are over expressed in certain tumor cells. For example, it is known that such kinases are frequently present in common human cancers such as breast cancer (Saimbury et al., Brit, J. Cancer, 1988, 58:458) and gastro intestinal cancers such colon, rectal and stomach cancers (Bolen et al., Oncogene Res., 1987, 1:149). It was discovered that Tyrosine Kinase activity (TK activity) is more frequently detectable in malignant cells than in normal cells (Hunter, Cell, 1987, 50:823).

More recently, it has been shown that Epidermal Growth Factor Receptor (EGFR) which possesses TK activity is over expressed in many human cancers such as brain, lung squamous cell, bladder, gastric, breast, head & neck, oesophageal, thyroid and the like. (W. J. Gullick, Brit. Med. Bull. 1991, 47:87). The receptor family includes four distinct members, including epidermal growth factor receptor (EGFR or ErbB1), HER2 (ErbB2 or p185^(neu)), HER3 (ErbB3) and HER4 (ErbB4 or tyro2). The HER (ErbB) family belongs to the subclass I receptor tyrosine kinase superfamily and consists of three distinct receptors, HER2, HER3, and HER4. Sequences of these receptors can be found in U.S. Pat. No. 5,183,884 (erbB3/HER3); U.S. Pat. No. 5,811,098 (HER4/Erb4 receptor); erbB2/HER2: Semba et al. (1985) Proc. Natl. Acad. Sci. USA 82:6497-6501 (designating the gene c-erbB-2); Coussens et al. (1985) Science 230:1132-1139 (designating the gene HER2); or King et al. (1985) Science 229:974-976.

Another receptor tyrosine kinase that is associated with cancer is the VEGF (vascular endothelial growth factor) receptor, the sequence of which is disclosed in U.S. Pat. No. 5,332,671.

A strategy to inhibit EGFR-TK activity has been exploiting small synthetic molecules (Arteaga C L, Exp. Cell Res., 2003, 284:122-130). Certain quinazoline derivatives like gefitinib (IRESSA®, Astra Zeneca), erlotinib (OSI-774, TARCEVA®), PD-183805, PKI-166, EKB-569, PD-168393, CGP-59362 have been have been investigated for possible treatment options for several forms of cancer (Baselga et al., Oncology 2002, 63: 6-16, Cohen R B., Clin. Colorectal Cancer, 2003, 2:246-251). The European patent applications namely EP 0566226, EP0602851A1, EP 0635507 A1, EP 0635498 A1, EO 0520722 A1 disclose certain quinazoline derivatives possessing anti-cancer properties and that inhibit TK.

U.S. Pat. No. 5,475,001, U.S. Pat. No. 5,457,105, U.S. Pat. No. 5,616,582, U.S. Pat. No. 5,770,599, U.S. Pat. No. 5,747,498, and U.S. Pat. No. 6,900,221 disclose quinazoline derivatives with structural features such as a substituted anilino moiety in the 4-position and a variety of functionalized alkyl groups in the 6- and 7-positions of the quinazoline nucleus. Specifically U.S. Pat. No. 5,457,105, U.S. Pat. No. 5,616,582 disclose N-(3-Chloro-4-fluorophenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine (Gefitinib) and U.S. Pat. No. 5,747,498 and U.S. Pat. No. 690,221 disclose N-(3-Ethylnylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine (Erlotinib). WO 20005/070909, WO 2007/060691 A₂ and WO 06/090413 disclose variations in synthesis or polymorphic forms of these two popular anti-cancer drugs.

Nonetheless, there remains a need for additional cancer therapies.

SUMMARY OF THE INVENTION

The present invention includes a method of treating drug resistant non-small cell lung cancer in a subject in need of treatment for drug resistant non-small cell lung cancer. The method includes administering to the subject a compound of formula (I):

in which:

R¹ is:

R² is —CH₃ or —CH₂CH₃; or a pharmaceutically acceptable salt of the compound of formula (I). Suitable pharmaceutically acceptable salts include a monohydrochloride, a dihydrochloride, or a mixture thereof. This method can employ a compound of formula (I) in which R¹ is:

and R² is —CH₃. This method can employ a compound of formula (IA):

The present invention includes the use of a compound of formula I or a pharmaceutically acceptable salt thereof for treating a subject suffering from drug resistant non-small cell lung cancer. The present invention also includes a use of a compound of formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a subject suffering from drug resistant non-small cell lung cancer.

The present invention includes a method of treating HER2 positive breast cancer in a subject in need of treatment for HER2 positive breast cancer. The method includes administering to the subject a compound of formula (I), which compound is described above, or a pharmaceutically acceptable salt thereof. The present invention includes the use of a compound of formula I or a pharmaceutically acceptable salt thereof for treating a subject suffering from HER2 positive breast cancer. The present invention also includes a use of a compound of formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a subject suffering from HER2 positive breast cancer.

The present invention includes a method of treating pancreatic cancer in a subject in need of treatment for HER2 positive breast cancer. The method includes administering to the subject a compound of formula (I), which compound is described above, or a pharmaceutically acceptable salt thereof. The present invention includes the use of a compound of formula I or a pharmaceutically acceptable salt thereof for treating a subject suffering from pancreatic cancer. The present invention also includes a use of a compound of formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a subject suffering from pancreatic cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph illustrating the results of experiments that compared the compound of formula IA to erlotinib and gemcitabine in causing tumor regression.

FIG. 2 shows results of immunohistochemical analysis of pancreatic sections (anti-angiogenic activity) for animals treated with the compound of formula IA, erlotinib and gemcitabine in causing tumor regression.

FIG. 3 is a graph illustrating the results of experiments that compared the compound of formula IA to lapatinib in causing tumor regression.

FIG. 4 is a graph illustrating the results of experiments that established regression of drug resistant tumors caused by the compound of formula IA.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “therapeutically effective dose” refers to a dose of the present compound of formula I (e.g., formula IA) that provides an amount of present compound in and around a tumor cell effective to achieve a desired biological activity of the present compound, e.g., reducing the rate of or stopping the growth of the tumor cell or killing the tumor cell. Desired biological activities include inhibiting EGFR tyrosine kinase of the tumor cell. Inhibiting the growth of a cell can include, for example, reducing the number of divisions the cell undergoes (e.g., to zero), killing the cell, reducing the population of a plurality of such cells, or the like.

As used herein, the term “effective amount” of the present compound of formula I (e.g., formula IA) is an amount sufficient to prevent, treat, reduce and/or ameliorate the symptoms and/or underlying causes of cancer or growth of a tumor cell. In some instances, an “effective amount” is sufficient to eliminate the symptoms of cancer or the growth of the tumor cell and, perhaps, overcome the cancer (e.g., put the cancer into remission) or the growth of the tumor cell. In the context of the present invention, the terms “treat” and “therapy” and the like refer to alleviate, slow the progression, prophylaxis, attenuation or cure of existing disease (e.g., cancer or growth of a tumor cell). Prevent, as used herein, refers to putting off, delaying, slowing, inhibiting, or otherwise stopping, reducing or ameliorating the onset of such diseases or disorders (e.g., cancer or growth of a tumor cell). It is preferred that a large enough quantity of the agent be dosed in non-toxic levels in order to provide an effective level of activity within the subject against the disease. The method of the present invention may be used with any mammal. Suitable mammals include, but are not limited to rats, cats, dogs, horses, cows, sheep, pigs, and, preferably, humans.

The Present Methods and Uses

The present invention includes a variety of methods employing the compound of the present invention. In an embodiment, the present method includes a method of inhibiting the growth of a tumor cell in a subject in need thereof. The tumor cell can be a pancreatic cancer cell, a HER2 positive breast cancer cell, or a drug (e.g., erlotinib or gefitinib) resistant non-small cell lung cancer cell. The present invention includes the use of a compound of formula I or a pharmaceutically acceptable salt thereof for treating a subject suffering from growth of a tumor cell, such as pancreatic cancer cell, a HER2 positive breast cancer cell, or a drug (e.g., erlotinib or gefitinib) resistant non-small cell lung cancer cell. These embodiments can include administering to the subject an effective amount of a compound of formula (I), which compound is described above, or a pharmaceutically acceptable salt thereof. These embodiments can employ a compound of formula (I) in which R¹ and R², are as described in embodiments above. These embodiments can employ a compound of formula (IA), which compound is described above, or a pharmaceutically acceptable salt thereof. These embodiments can employ a pharmaceutical composition including a compound of formula (I) or a pharmaceutically acceptable salt thereof. In an embodiment, the tumor cell is a pancreatic cancer cell. In an embodiment, the tumor cell is a HER2 positive breast cancer cell. In an embodiment, the tumor cell is a drug resistant non-small cell lung cancer cell. In an embodiment, the drug is erlotinib, gefitinib, or a plurality thereof.

The present invention also includes a use of a compound of formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a subject suffering from growth of a tumor cell, such as pancreatic cancer cell, a HER2 positive breast cancer cell, or a drug (e.g., erlotinib or gefitinib) resistant non-small cell lung cancer cell. This embodiment can employ a compound of formula (I) in which R¹ and R², are as described in embodiments above. This embodiment can employ a compound of formula (IA), which compound is described above, or a pharmaceutically acceptable salt thereof.

In an embodiment, the present method includes a method of treating drug resistant non-small cell lung cancer in a subject in need of treatment for drug resistant non-small cell lung cancer. The present method also includes the use of a compound of formula I for treating a subject suffering from drug resistant non-small cell lung cancer. In an embodiment, the drug is erlotinib, gefitinib, or a plurality thereof. These embodiments of the method can include administering to the subject an effective amount of a compound of formula (I), which compound is described above, or a pharmaceutically acceptable salt thereof. These embodiments of the method can employ a compound of formula (I) in which R¹ and R², are as described in embodiments above. These embodiments of the method can employ a compound of formula (IA), which compound is described above, or a pharmaceutically acceptable salt thereof.

The present invention also includes a use of a compound of formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a subject suffering from drug (e.g., erlotinib or gefitinib) resistant non-small cell lung cancer. This embodiment can employ a compound of formula (I) in which R¹ and R², are as described in embodiments above. This embodiment can employ a compound of formula (IA), which compound is described above, or a pharmaceutically acceptable salt thereof.

In an embodiment, the present method includes a method of treating HER2 positive breast cancer in a subject in need of treatment for HER2 positive breast cancer. The present method also includes the use of a compound of formula I for treating a subject suffering from HER2 positive breast cancer. These embodiments of the method can include administering to the subject an effective amount of a compound of formula (I), which compound is described above, or a pharmaceutically acceptable salt thereof. These embodiments of the method can employ a compound of formula (I) in which R¹ and R², are as described in embodiments above. These embodiments of the method can employ a compound of formula (IA), which compound is described above, or a pharmaceutically acceptable salt thereof.

The present invention also includes a use of a compound of formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a subject suffering from HER2 positive breast cancer. This embodiment can employ a compound of formula (I) in which R¹ and R², are as described in embodiments above. This embodiment can employ a compound of formula (IA), which compound is described above, or a pharmaceutically acceptable salt thereof.

In an embodiment, the present method includes a method of treating pancreatic cancer in a subject in need of treatment for pancreatic cancer. The present method also includes the use of a compound of formula I for treating a subject suffering from pancreatic cancer. These embodiments of the method can include administering to the subject an effective amount of a compound of formula (I), which compound is described above, or a pharmaceutically acceptable salt thereof. These embodiments of the method can employ a compound of formula (I) in which R¹ and R², are as described in embodiments above. These embodiments of the method can employ a compound of formula (IA), which compound is described above, or a pharmaceutically acceptable salt thereof.

The present invention also includes a use of a compound of formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a subject suffering from pancreatic cancer. This embodiment can employ a compound of formula (I) in which R¹ and R², are as described in embodiments above. This embodiment can employ a compound of formula (IA), which compound is described above, or a pharmaceutically acceptable salt thereof.

Unexpected Advantages of the Compounds of the Present Invention

Compounds of the present invention exhibit unexpected advantages in methods of the present invention. For example, compounds of the present invention (e.g., a compound of formula (IA)) has exhibited unexpectedly superior anti-cancer and anti-proliferative effects compared to known compounds such as erlotinib, gefitinib, and lapatinib.

The compounds of the present invention (e.g., a compound of formula (IA)) exhibited activity against numerous kinases in in vitro assays. The compound of formula (IA) was evaluated for inhibition of 80 kinases in an in vitro kinase profiling study and significantly inhibited 15 of them.

The compound of formula (IA) was evaluated in an in vivo model of pancreatic cancer and found to be unexpectedly superior to erlotinib alone or in combination with gemcitabine. The compound of formula (IA) caused regression of subcutaneous pancreatic tumors in nude mice. The tumor regression observed was superior to that caused by the existing drugs erlotinib and gemcitabine.

The compound of formula (IA) was evaluated in an in vivo model of HER2 positive breast cancer. The tumor regression observed with the compound of formula (IA) in SCID mouse xenograft models of HER2 positive breast cancer was superior to that of Lapatinib, an approved drug of choice for treatment of HER2 positive breast cancer.

The compound of formula (IA) was evaluated in an in vivo model of drug (e.g., erlotinib or Gefitinib) resistant non-small cell lung cancer. The compound of formula (IA) showed significant anticancer activity in animals implanted with erlotinib/gefitinib resistant H1975 cells. Tumor regression was observed after withdrawal of treatment with the compound of formula (IA).

Embodiments of the Present Methods

In an embodiment, a compound of the present invention can be employed to inhibit the growth of a cell including a tyrosine kinase. For example, a compound of the present invention can inhibit the growth of a tumor cell or malignant cell (e.g., a pancreatic cancer cell, a HER2 positive breast cancer cell, or a drug (e.g., erlotinib or gefitinib) resistant non-small cell lung cancer cell) that expresses a tyrosine kinase, such as an epidermal growth factor (EGF) receptor tyrosine kinase, an Erb-2 tyrosine kinase, an Erb-3 tyrosine kinase, an Erb-4 tyrosine kinase, or a VEGF receptor tyrosine kinase. Such a tumor cell or malignant cell can over express the receptor tyrosine kinase. In an embodiment, the cell is a drug (e.g., erlotinib or gefitinib) resistant non-small cell lung cancer cell. In an embodiment, the cancer cell is a pancreatic cancer cell. In an embodiment, the cancer cell is a breast cancer cell that expresses HER2. In certain embodiments, the present compound can inhibit the growth of the tumor cell or malignant cell in vitro, ex vivo, or in vivo.

In an embodiment, the method can include contacting a cell expressing the tyrosine kinase with a compound of the present invention. In an embodiment, the method can include contacting a tumor containing a cell expressing the tyrosine kinase with a compound of the present invention. In certain embodiments, the present method can include contacting in vitro, ex vivo, or in vivo.

In an embodiment, the method can include contacting a tumor cell or malignant cell (e.g., a pancreatic cancer cell, a HER2 positive breast cancer cell, or a drug (e.g., erlotinib or gefitinib) resistant non-small cell lung cancer cell) that expresses a tyrosine kinase, such as an epidermal growth factor (EGF) receptor tyrosine kinase, an Erb-2 tyrosine kinase, an Erb-3 tyrosine kinase, an Erb-4 tyrosine kinase, or a VEGF receptor tyrosine kinase with a compound of the present invention. In certain embodiments, the method can include contacting a pancreatic cancer cell, a HER2 positive breast cancer cell, or a drug resistant (e.g., erlotinib or gefitinib resistant) non-small cell lung cancer cell with a compound of the present invention. In an embodiment, the method can include contacting a drug resistant (e.g., erlotinib or gefitinib resistant) non-small cell lung cancer cell with a compound of the present invention. In an embodiment, the method can include contacting a pancreatic cancer cell with a compound of the present invention. In an embodiment, the method can include contacting a breast cancer cell that expresses HER2 with a compound of the present invention. In certain embodiments, the present method can include contacting in vitro, ex vivo, or in vivo.

In an embodiment, the method can include contacting a tumor (e.g., a pancreatic cancer tumor, a HER2 positive breast cancer tumor, or a drug (e.g., erlotinib or gefitinib) resistant non-small cell lung cancer tumor) that expresses a tyrosine kinase, such as an epidermal growth factor (EGF) receptor tyrosine kinase, an Erb-2 tyrosine kinase, an Erb-3 tyrosine kinase, an Erb-4 tyrosine kinase, or a VEGF receptor tyrosine kinase with a compound of the present invention. In certain embodiments, the method can include contacting a pancreatic cancer tumor, or a HER2 positive breast cancer tumor, or a drug (e.g., erlotinib or gefitinib) resistant non-small cell lung cancer tumor with a compound of the present invention. In an embodiment, the method can include contacting a drug resistant (e.g., erlotinib or gefitinib resistant) non-small cell lung cancer tumor with a compound of the present invention. In an embodiment, the method can include contacting a pancreatic cancer tumor with a compound of the present invention. In an embodiment, the method can include contacting a breast cancer tumor that expresses HER2 with a compound of the present invention. In certain embodiments, the present method can include contacting in vitro, ex vivo, or in vivo.

In an embodiment, the method can include administering the compound of the present invention to a subject (e.g., a mammal or a warm blooded animal) having a tumor containing a cell (e.g., a pancreatic cancer cell, a HER2 positive breast cancer cell, or a drug (e.g., erlotinib or gefitinib) resistant non-small cell lung cancer cell) expressing the tyrosine kinase. In an embodiment, the method can include administering the compound of the present invention to a subject (e.g., a mammal or a warm blooded animal) having a tumor cell or malignant cell (e.g., a pancreatic cancer cell, a HER2 positive breast cancer cell, or a drug (e.g., erlotinib or gefitinib) resistant non-small cell lung cancer cell) that expresses a tyrosine kinase, such as an epidermal growth factor (EGF) receptor tyrosine kinase, an Erb-2 tyrosine kinase, an Erb-3 tyrosine kinase, an Erb-4 tyrosine kinase, or a VEGF receptor tyrosine kinase. In certain embodiments, the method can include administering the compound of the present invention to a subject (e.g., a mammal or a warm blooded animal) having pancreatic cancer, HER2 positive breast cancer, or drug resistant (e.g., erlotinib or gefitinib resistant) non-small cell lung cancer. In an embodiment, the method can include administering the compound of the present invention to a subject (e.g., a mammal or a warm blooded animal) having drug resistant (e.g., erlotinib or gefitinib resistant) non-small cell lung cancer. In an embodiment, the method can include administering the compound of the present invention to a subject (e.g., a mammal or a warm blooded animal) having pancreatic cancer. In an embodiment, the method can include administering the compound of the present invention to a subject (e.g., a mammal or a warm blooded animal) having breast cancer that expresses HER-2. In an embodiment, the subject is a human.

The methods have been described with reference to “a compound of the present invention.” In the description of the methods of the invention, the phrase “a compound of the present invention” can be replaced by the phrase “a pharmaceutically acceptable salt of a compound of the present invention”, the phrase “a pharmaceutical composition including a compound of the present invention” or the phrase “a pharmaceutical composition including a pharmaceutically acceptable salt of a compound of the present invention” to describe additional embodiments of the present methods.

The compound of the present invention can be administered at a dose sufficient to provide a therapeutically effective level at a tumor cell in the subject. It is recognized that the total amount of compound of the present invention administered as a unit dose to a subject will depend upon the type of pharmaceutical composition being administered. It should be apparent to a person skilled in the art that variations may be acceptable with respect to the therapeutically effective dose and frequency of the administration of compound of the present invention in this embodiment of the invention. The amount of the compound of the present invention administered will be inversely correlated with the frequency of administration. Hence, an increase in the concentration of compound of the present invention in a single administered dose, or an increase in the mean residence time in the case of a sustained release form of compound of the present invention, generally will be coupled with a decrease in the frequency of administration.

The actual dose of the compound of the present invention will depend on a variety of factors that may be specific to the subject undergoing dosing. These factors should be taken into consideration when determining the therapeutically effective dose of compound of the present invention and frequency of its administration. For example, the effective dose can depend on the species, age, weight, or general health of the subject; the severity of the cancer or tumor growth; the size and location of the tumor in which an effective amount of agent must be achieved; the frequency and duration of dosing; the type of formulation administered; and the like. Generally, a higher dosage is preferred if the disease or disorder is more severe.

Methods of Making the Present Compounds

Scheme 1 illustrates an embodiment of a process for making compounds of the present invention.

Pharmaceutical Compositions

The present compound can be in a pharmaceutical composition that includes a pharmaceutically acceptable carrier mixed with the compound of the present invention and other components in the pharmaceutical composition. As used herein, the term “pharmaceutically acceptable carrier” refers to a carrier that is conventionally used to facilitate the storage, administration, and/or the healing effect of the an anticancer or antiproliferative agent. A carrier may also reduce any undesirable side effects of the agent. A suitable carrier should be stable, i.e., incapable of reacting with other ingredients in the formulation. It should not produce significant local or systemic adverse effect in recipients at the dosages and concentrations employed for treatment. Such carriers are generally known in the art.

In an embodiment, the pharmaceutical composition can include:

Tablet mg/tablet Compound NRC-2694 or a salt thereof 50 Lactose anhydrous (USP) 156 Microcrystalline cellulose (Avicel pH102) 15 Sodium lauryl sulfate 5 Sodium starch glycolite 10 Povidone K-30 3 Hydroxy propyl cellulose (LH-11) 10 Magnesium stearate 1

The agent of the present invention can also be formulated in a sustained-release form to prolong the presence of the pharmaceutically active agent in the treated mammal, generally for longer than one day. Many methods of preparation of a sustained-release formulation are known and are disclosed in Remington's Pharmaceutical Sciences (18th ed.; Mack Publishing Company, Eaton, Pa., 1990).

Articles and Methods of Manufacture

The present invention also includes an article of manufacture providing an agent for administration to a subject having a tumor or to a tumor cell. The article of manufacture can include a container which contains a composition (e.g., tablet) suitable for the present method. The article of manufacture further includes instructions in the form of a label on the container and/or in the form of an insert included in a box in which the container is packaged, for the carrying out the method of the invention. The instructions can also be printed on the box in which the container is packaged. The instructions contain information such as sufficient dosage and administration information so as to allow the subject or a worker in the field to administer the present compound or pharmaceutical composition. It is anticipated that a worker in the field encompasses any doctor, nurse, technician, spouse, or other caregiver who might administer the agent. The agent can also be self-administered by the subject.

According to the invention, the present compound of formula I (e.g., compound of formula IA) can be used for manufacturing an composition or medicament including the compound and suitable for oral or parenteral administration. The invention also relates to methods for manufacturing a composition or medicament including a compound of formula I (e.g., compound of formula IA) suitable for oral or parenteral administration. For example, a tablet can be manufactured in several ways, using conventional techniques. A liquid composition can be manufactured by dissolving an agent in a suitable solvent, such as water, at an appropriate pH, including buffers or other excipients, for example, to form a solution.

The present invention may be better understood with reference to the following examples. These examples are intended to be representative of specific embodiments of the invention, and are not intended as limiting the scope of the invention.

EXAMPLES Example 1 Preparation of N-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine (IA) i) Preparation of 4-Chloro-6-[3-(4-morpholinyl)propoxy-4-quinazoline (IIIa)

Into a clean and dried 5-Litre four necked round bottomed flask equipped with a mechanical stirrer, reflux-condenser, pressure equalizing addition funnel, and thermometer socket were charged chloroform (3000 ml), dimethyl formamide (30 ml) followed by 7-methoxy-6-(3-morpholino propoxy)-3,4-dihydro-quinazolin-4-one (IIa) (150 g), obtained according to the process given in Example-1 of PCT international application published as WO2005/070909A1. Oxalyl Chloride (120 g) was slowly added and the reaction mass was heated to reflux temperature and maintained at reflux temperature for about 5 hours. Reaction was found to be completed by HPLC test. The solvent chloroform and excess oxalyl chloride were distilled off by applying mild vacuum. The reaction mass was cooled to about 40° C. and added chloroform (300 ml) and again distilled out the solvent by applying mild vacuum. The reaction mixture was cooled to room temperature and acetonitrile (3000 ml) was added and stirred for 10-15 minutes and kept under nitrogen atmosphere to proceed to the next step.

ii) Preparation of N-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine (IA)

Into a 5-Litre four necked round bottomed flask equipped with a mechanical stirrer, reflux-condenser and thermometer socket containing the chloro compound in acetonitrile from the above step-(i); 3-ethynyl aniline (69 g) was added slowly in about 10-15 minutes and the reaction mass was heated to reflux temperature and maintained at reflux temperature for about 4 hrs. The reaction was found to be completed by HPLC test. Then the reaction mass was cooled to 25-35° C. and filtered, washed the cake with acetonitrile (500 ml) and dried the cake.

The above dried crude compound was taken into a another 5 liter round bottomed flask and charged water (2500 ml) and slowly raised the temperature to 60-65° C. and was adjusted the pH of the reaction mass to 10-12 with dilute sodium hydroxide solution. The solid product separated was filtered and washed with water and dried at 70-75° C. to get 173.0 g of N-(3-ethynylphenyl)-6-(3-morphiline propoxy)-7-methoxy-4-quinazolamine as a off-white solid.

iii) Recrystallisation of Preparation of N-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine from Toluene.

Into a 5-Litre four necked round bottomed flask equipped with a mechanical stirrer, reflux-condenser and thermometer socket were charged toluene (3750 ml), followed by N-(3-ethynylphenyl)-6-(3-morpholino propoxy)-7-methoxy-4-quinazolinamine (50 g) obtained by the process described in the above given example-(1). The reaction mixture was heated to 90-95° C., so that the solid completely dissolved. Then carbon treatment was given and filtered. The filtrate was cooled to 25-35° C., maintained for about 1 hour and filtered and dried the material to get 40.15 g of N-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine as a white crystalline solid.

mp: 185-187° C.

Purity: 99.72% (HPLC)

IR (KBr) (cm⁻¹): 3280.9, 2954.6, 2810.3, 1620.1, 1604.2, 1572.1, 1527.7, 1505.2, 1484, 1430.5, 1388.2, 1247.5, 1211.2, 1140.3, 1110.4, 1010.3, 953.4, 859.6, 784.2 Cm⁻¹

¹HNMR (300 MH_(z); DMSO-d₆): 9.57 (s, 1H); 8.48 (s, 1H); 7.99 (s, 1H); 7.86 to 7.92 (d, 2H); 7.34 to 7.44 (t, 1H) 7.18 to 7.21 (s, 2H); 4.15 to 4.21 (t, 4H); 3.92 (s, 3H) 3.5 to 3.6 (t, 4H); 2.4 to 2.52 (m, 5H); 1.95 to 2.01 (m, 2H).

Mass: 419.4 (M+1)

Example 2 Recrystallisation of N-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine from acetonitrile

Into a two liter three necked round bottomed flask equipped with a mechanical stirrer, reflux-condenser and thermometer socket were charged acetonitrile (1000 ml), followed by N-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine (25 g) obtained from the process described in the above given Example-(1). The reaction mass was slowly heated to 65-70° C., so that the solid material completely dissolved and carbon treatment was given and filtered the reaction mass. The filtrate was transferred into another round-bottomed flask and slowly cooled to 10-15° C. and maintained for 30 minutes at that temperature. The mass was filtered and after washing the cake with chilled acetonitrile dried to get 20.50 g of N-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine as a white crystalline solid.

mp: 186-187° C.;

Purity: 99.68% (HPLC)

Example 3 Recrystallisation of N-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine from Ethyl acetate

Into a three liter three necked round bottomed flask equipped with a mechanical stirrer, reflux-condenser and thermometer socket were charged ethyl acetate (2000 ml), followed by N-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine (25 g) obtained from the process described in the above given Example-(1). The reaction mass was slowly heated to 65-70° C., so that the solid material completely dissolved and carbon treatment was given and filtered the reaction mass. The filtrate was transferred into another round-bottomed flask and slowly cooled to 10-15° C. and maintained for 30 minutes at that temperature. The crystalline mass was filtered and after washing the cake with chilled ethyl acetate dried to get 20.95 g of N-(3-ethynlphenyl)-6-(3-morpholino propoxy)-7-methoxy-4-quinazolinamine as a white crystalline solid.

mp: 185-187° C.

Purity: 99.7% (HPLC)

Example 4 Preparation of N-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine mono hydrochloride (IA monohydrochloride)

Into a 500 ml three necked round bottomed flask equipped with a mechanical stirrer, reflux-condenser, thermometer socket etc. charged Isopropyl alcohol (250 ml), followed by N-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine (5 g), obtained from the process given in Example-1. The temperature of the reaction mass was raised to 65-70° C. so that all the solid material dissolves and carbon treatment was given and filtered. The filtrate was cooled to about 55 to 60° C. and to this one mole equivalent of HCl-gas dissolved in isopropyl alcohol solution was added when the mono hydrochloride salt separated out. The reaction mass was maintained at reflux temperature for about 2 hrs and then cooled to room temperature and filtered and dried to get 5.1 g. of N-(3-ethynyl phenyl)-6-(3-morpholino propoxy)-7-methoxy-4-quinazolinamine mono hydrochloride as a white crystalline substance.

Purity: 99.8% (HPLC)

HCl content (chemical): 8.19% (Theoretical value: 8.01%)

IR (KBr) (cm⁻¹): 3407, 3305, 3259.5, 2934, 2619, 1625.9, 1593.8, 1579.9, 1530.8, 1512, 1476.9, 1392.2, 1356.8, 1282.1, 1242.1, 1207.9, 1141.3, 1100.8, 1076.1, 1042.1, 1026.5, 1011.5, 957.7, 941.5, 922.1, 857.3, 852, 838.1, 796, 782.4.

Example 5 Preparation of N-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine di hydrochloride (IA dihydrochloride)

Into a 500 ml three necked round bottomed flask equipped with a mechanical stirrer, reflux-condenser and thermometer socket were charged Isopropyl alcohol (250 ml), followed by N-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine 5 g), obtained from the process given in Example-1. The temperature of the reaction mass was raised to 65-70° C. so that all the solid material dissolves. Carbon treatment was given and filtered. The filtrate was cooled to about 55 to 60° C. and to this two moles equivalent of HCl-gas dissolved in isopropyl alcohol solution was added when the dihydrochloride salt separated out. The reaction mass was maintained at reflux temperature for about 2 hrs and then cooled to room temperature and filtered and dried to get 5.5 g. of N-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine di hydrochloride as a white crystalline substance.

Purity: 99.78% (HPLC)

HCl content (chemical): 14.9% (Theoretical value: 14.83%)

IR (KBr) (cm⁻¹): 3406.8, 3194.1, 2942.7, 2681.9, 2623.6, 1633.7, 1566.2, 1528.6, 1512.5, 1438.6, 1359.6, 1282.3, 1218.3, 1157.1, 1132.7, 1105.9, 1075.6, 1001.9, 942.1, 875.3, 816.1, 787.2

Example 6 In Vitro Kinase Profiling of the Compound of Formula (IA)

NRC-2694 (the compound of formula IA) was found to possess activity various kinases from a panel of 80 kinases by the method of KinomeScan™ of Ambit Biosciences Corp, San Diego, Calif. The kinases were ABL1(F317L), ABL1(H396P), ABL1(Q252H), ABL1(Y253F), ADCK3, ADCK4, ALK, ARK5, AXL, BLK, BRSK2, CDK7, CIT, CLK4, CSNK1D, CSNK1E, DAPK1, DAPK3, DCAMKL3, DMPK, EGFR, EGFR(E746-A750DEL), EGFR(G719C), EGFR(G719S), EGFR(L747-E749DEL, A750P), EGFR(L747-S752 DEL, P753S), EGFR(L747-T751DEL, Sin), EGFR(L858R), EGFR(L861Q), EGFR(S752-I759DEL), EPHA5, EPHA6, ERBB2, ERBB4, ERK3, ERK4, FGR, FLT3(D835H), FLT3(D835Y), FLT3(ITD), FLT3(N841 I), FRK, GAK, GCN2(KIN.DOM.2, S808G), HCK, IRAK3, JAK1(KIN.DOM.1), KIT, KIT(D816V), KIT(V559D), KIT(V559D,T670I), LCK, LOK, LTK, LYN, MAP3K3, MAP4K4, MAP4K5, MET, MINK, MKNK1, MKNK2, MST4, MYLK2, PDGFRB, PIM3, PKN2, PRKD1, PRKD2, PRKD3, PRKG1, RIPK2, RPS6KA4(KIN.DOM.2), SIK2, SLK, SNARK, SRC, STK36, TNIK, TNNI3K, TYK2(KIN.DOM.2).

Kinase assays: Assays were performed as described in Fabian et al. (2005) Nature Biotechnology, vol. 23, p. 329. Kinase-tagged T7 phage strains were grown in parallel in 24 or 96-well blocks in an E. coli host derived from the BL21 strain. E. coli were grown to log-phase and infected with T7 phage from a frozen stock (multiplicity of infection ˜0.1) and incubated with shaking at 32° C. until lysis (˜90 minutes). The lysates were centrifuged and filtered to remove cell debris. Streptavidin-coated magnetic beads were treated with biotinylated small molecule ligands for 30 minutes at room temperature to generate affinity resins for kinase assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific phage binding. Binding reactions were assembled by combining phage lysates, liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT).

Test compounds were prepared as 40× stocks in DMSO and diluted into the aqueous environment. 2.5% DMSO was added to control assays lacking a test compound. All reactions were performed in polystyrene 96-well plates that had been pretreated with blocking buffer in a final volume of 0.04 ml. The assay plates were incubated at room temperature with shaking for 1 hour, long enough for binding reactions to reach equilibrium, and the affinity beads were washed four times with wash buffer (1×PBS, 0.05% Tween 20, 1 mM DTT) to remove unbound phage. After the final wash, the beads were resuspended in elution buffer (1×PBS, 0.05% Tween 20, 2 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The phage titer in the eluates was measured by quantitative PCR.

Results

The activity profile results are shown in charts 1.1-1.6. Significant values are shaded. The kinases against which NRC-2694 (the compound of formula IA) is most active is indicated as (++) and where it has shown very little inhibiting effect is indicated as (−−) in the following table.

Kinase Activity Trends-NRC2694 (IA) (++) (−−) ALK, AKT1, AKT2, BLK, ASK1, AURKA₁, CDK7, BMPR¹A, BMPR², CIT CDK5, CDK8, EGFR EPHA2, ERK2, ERK-3 FGFR², FGFR3, ERK-4 GSK3B, LMK2, FLT MARK4, GAK, MEK3, MLK1, MST1, LCK MUSK, MYO3B, LOK p38, PCTK, P1K3CA, MKNK PKAC, PRKX, PDGFR-B RAF-1, RET, RPS6KA, PRKD SRMS, STK16, SLK TRKA, ULK, RIPK2 YANK, ZAP70

Example 7 Anticancer Activity of the Compound of Formula (IA) in Pancreatic Cancer Models

In vivo anticancer activity of NRC-2694 (the compound of formula IA) for the treatment of Pancreatic Cancer in nude mice xenografts (Athymic nude mice).

Methodology

Athymic nude mice were implanted with 10×10⁶ pancreatic cancer cells MIA PaCa-2 (ATCC #CRL1420). After tumor formation (˜0.3-0.05 cm³) mice were treated with study compounds with or without Gemcitabine. Tumor volume and animal activity were measured at regular intervals. At the termination of the experiment tumors were collected and fixed in 10% buffered formaldehyde for 12 hours and processed for paraffin embedding followed by sectioning. Tumor sections were immunoprobed for VEGF and Ki67 and percent expression determined with respect to controls.

Study Design Group # Study Drugs Dose (mg/kg) Route Number of Animals 1 Control 0 Oral 5 Male + 5 Females 2 NRC 2694 20 Oral 5 Male + 5 Females 3 Erlotinib 50 Oral 5 Male + 5 Females 4 Gemcitabine 120 i.p. 5 Male + 5 Females 5 NRC 2694 + 20 + 120 Oral + ip 5 Male + 5 Females Gemcitabine 6 Erlotinib + 50 + 120 Oral + ip 5 Male + 5 Females Gemcitabine

Results

From the study conducted it was observed that NRC-2694 (the compound of formula IA) did cause the regression of pre-established subcutaneous pancreatic tumors in nude mice. Tumor regression caused by NRC2694 (the compound of formula IA) was similar to Erlotinib and Gemcitabine treatments. However, addition of Gemcitabine caused a greater than additive effect. Erlotinib hydrochloride (TARCEVA®) is an approved standard drug for the first-line treatment of patients with locally advanced, unresectable or metastatic pancreatic cancer, in combination with Gemcitabine. Gemcitabine (GEMZAR®) is indicated for the first line treatment for patients with locally advanced non-resectable or metastatic adenocarcinoma of the pancreas.

The tumor regression caused by treating with compound IA was superior to that caused by Erlotinib and Gemcitabine individually as well as in combination (FIG. 1). This observation was further confirmed in Immunohistochemical analysis (FIG. 2).

Example 8 Anticancer Activity of the Compound of Formula (IA) in HER-2 Positive Breast Cancer

In vivo anticancer activity of NRC-2694 (the compound of formula IA) for the treatment of HER-2 Positive Breast Cancer in SCID mice xenografts.

Methodology

Animals were subcutaneously implanted with 2×10⁶ cells. After the development of observable tumors drug treatment was initiated. As per IRB regulations no animals were allowed to bear tumors greater than 15 mm. Cell line: BT474/HTB20 human breast cancer cell line.

Study Design Group # Study Drugs Dose (mg/kg) Route Number of Animals 1 Control 0 Oral 5 Male + 5 Females 2 NRC 2694 10, 20 and 40 Oral 5 Male + 5 Females (once a day) 3 Lapatinib 30 and 100 Oral 5 Male + 5 Females (Twice a day)

Results

NRC-2694 (the compound of formula IA) demonstrated a dose dependent increase in anti tumor activity in HER-2 positive Breast cancer model. The tumor regression observed with NRC-2694 (the compound of formula IA) treated animals was superior to that of Lapatinib treated animals (FIG. 3). Lapatinib (TYKERB®) is a kinase inhibitor indicated in combination with Capecitabine for the treatment of patients with advanced or metastatic breast cancer whose tumors over express HER2 and who have received prior therapy including an anthracycline, a taxane and trastuzumab. NRC-2694 (the compound of formula IA) was superior to Lapatinib, the established treatment option for HER2 expressing tumors, which was significant and unexpected.

Example 9 Anticancer Activity of the Compound of Formula (IA) in Erlotinib/Gefitinib Resistant NSCLC Model

In vivo activity of NRC-2694 (the compound of formula IA) for the treatment of Erlotinib/Gefitinib resistant Non Small Cell Lung Cancer (NSCLC) in nude mice xenografts (athymic nude mice).

Methodology

Cells: H1975. Athymic nude mice, 5-8 weeks of age and weighing 20 to 25 g, were housed in the animal facility. H1975 cells were harvested from exponentially growing cultures, detached by brief trypsinization, washed twice in cold HBSS, resuspended in ice-cold HBSS, and implanted subcutaneously (3×10⁶ cells per mouse) into the dorsal hind flank. Treatment with the study drugs was initiated only after the tumor volume reached to 200-300 mm³.

Study Design Study Dose Group # Drugs (mg/kg) Route Number of Animals 1 Control 0 Oral 5 Male + 5 Females 5 NRC 2694 10 Oral 5 Male + 5 Females 6 NRC 2694 20 Oral 5 Male + 5 Females 7 NRC 2694 40 Oral 5 Male + 5 Females 8 Erlotinib 100 Oral 5 Male + 5 Females

Results

NRC 2694 (the compound of formula IA) demonstrated significant anticancer activity in animals implanted with Erlotinib/Gefitinib NSCLC resistant cells compared to Erlotinib or control. It showed dose dependent decrease in the tumor volume. Tumor remission was continued in animals treated with NRC 2694 (the compound of formula IA) even after withdrawing the study treatment (FIG. 4).

Drug resistance for treatment of non-small cell lung cancer (NSCLC) by approved drugs like Erlotinib (TARCEVA®) and Gefitinib (IRESSA®) is a clinical challenge posed and observed in recent times (Clin. Cancer. Res. 2006, 12(19): 5764-69; PLOS Medicine, March 2005, Online edition 0225-0235). The significant superior activity (250% reduction in tumor volume) observed with NRC-2694 (the compound of formula IA) was unexpected.

Example 10 NRC-2694 is Less Toxic Than Erlotinib in Swiss Albino Mice

This study determined that NRC-2694 (the compound of formula IA) is less toxic than erlotinib in Swiss albino mice. The maximum tolerated dose (MTD) of NRC-2694 is about twice that of erlotinib. The MTD of NRC-2694 is the same in mice and rats.

Materials and Methods

The method followed was as per the guidelines of FDA (Aug. 26, 1996).

Swiss Albino Mice

20 male and 20 female Swiss albino mice were randomly divided into four groups (G1-G4). Each group was 5 male and 5 female Swiss albino mice. Groups G2, G3 and G4 were treated with NRC-2694 monohydrochloride through oral route at the dose levels of 250, 500 and 1000 mg/kg body weight respectively. G1 was treated with placebo and served as a control. The oral formulation included 25 mg/mL of NRC-2694 that was administered in doses of 10 mL/kg body weight (b.wt) to each animal. Animals from the low dose group (G2-treated with 250 mg/kg b.wt) were administered the oral formulation as a single dose. The mid dose group animals (G3-treated with 500 mg/kg b.wt) were administered the dose formulation twice with a gap of approximately 1 hour. Animals from high dose group (G4-treated with 1000 mg/kg b.wt) received the dose formulation four times with a gap of approximately one hour.

Dose formulation analysis for test substance revealed 105.1, 105.74 and 105.76% recovery whereas dose formulation of placebo substance revealed 0% recovery of active ingredient.

At the end of observation period of 14 days, all surviving mice were sacrificed and subjected to gross pathological examinations.

Wistar Rats

20 male and 20 female Wistar rats were randomly divided in to four groups and dosed as described above for the mice. Dose formulation analysis for test substance revealed 101.0, 102.84 and 103.56% recovery whereas dose formulation of placebo substance revealed 0% recovery of active ingredient.

Results

Mice: No mortality was observed in mice from the control as well as the treatment groups. Clinical signs of toxicity such as mild lethargy and piloerection were observed in high dose group after dosing during days 10 to 14. No significant alterations were observed in mean body weight and percent body weight when compared with their respective control groups. Gross pathological examination after day 14 did not reveal any lesions of pathological significance.

Rats: No mortality was observed in rats from the control as well as the treatment groups. In the high dose treated group (G4-1000 mg/kg body wt.) clinical signs of toxicity such as mild to severe lethargy (10/10), piloerection 10/10), chromodacryoorhea (10/10), weakness (1/10) and ptosis (4/10) were observed. Statistically significant reduction was observed in body weight and percent body weight change of mid and high dose treated group of rats on days 7 and 14. Reduction observed in the body weight and percent body weight change in high dose treated animals was considered as an effect of oral administration of NRC-2694 monohydrochloride. Gross pathological examination did not reveal any lesions of pathological significance, except minimal uterus distension in two female rats. This lesion was physiological or cyclic in nature and could be considered as a spontaneous finding.

Conclusions

Mice: Based on the findings of this study, it was concluded that the test substance NRC-2694 monohydrochloride did not produce any toxic signs or mortality when administered through oral gavage at the dose level of 250 mg/kg body weight (low dose) and 500 mg/kg (mid dose). At 1000 mg/kg (high dose) the NRC-2694A blend produced signs of lethargy and piloerection in all animals without any mortality. The MTD of NRC-2694 monohydrochloride by acute oral gavage in Swiss albino mice was 1000 mg/kg body weight under the condition and the procedures followed in the present study.

Under our laboratory condition, the MTD of Erlotinib HCl was recorded to be 500 mg/kg, when given orally to Swiss albino mice. Thus, NRC-2694 is advantageously significantly less toxic than Erlotinib HCl in mice.

Rats: Based on the findings of this study, it was concluded that the test substance NRC-2694A blend did not produce any major toxic signs or mortality when administered through oral gavage at the dose level of 250 mg/kg body weight (low dose) and 500 mg/kg body weight (mid dose). However, at the dose level of 1000 mg/kg body weight (high dose), the NRC-2694A blend produced marked toxic signs with reduction in body weight but devoid of any pathological lesions and mortality. Based on the above study, the MTD of NRC-2694A blend by acute oral gavage in Wistar rats is considered as 1000 mg/kg body weight under the condition and the procedures followed in the present study.

Example 11 NRC-2694 is Less Toxic Than Erlotinib in Beagle Dogs

This study determined that NRC-2694 (the compound of formula IA) is less toxic than erlotinib in beagle dogs. The no observed adverse effect level (NOAEL) of NRC-2694 is about twice that of erlotinib.

Materials and Methods

The methods followed were as per CPMP/SWP/1042/99 (July 2000) and ICH S3A (March (1995)) guidelines. Prior to the 30 day repeated dose study, a dose range finding study was conducted, which included single dose MTD study followed by a 10 day repeated dose study. For the single dose MTD study, two dogs (1 male and 1 female) of beagle breed, obtained from Marshall's Farm, China, were used. The route of dosing was oral, by gelatin capsule. Based on the results of the dose range finding study, doses for the 30 days study were selected.

The 30 day repeated dose study employed 12 male and 12 female dogs (Marshall's Farm, China). The dogs were acclimatized for a minimum period of 2 weeks and randomly divided into four main groups (G1-G4), each group comprising of 3 dogs per sex. The animals were dosed orally with capsules of NRC-2694A for a period of 30 consecutive days at dose levels of 10 mg/kg b.wt/day (G2-low dose), 20 mg/kg b.wt/day (G3-mid dose) and 40 mg/kg b.wt/day (G4-high dose). However, the high dose was subsequently decreased to 30 mg/kg b.wt/day. The control group (G1-0 mg/kg b. wt/day) animals were dosed by oral capsule with placebo.

Each dog was observed for visible signs of reaction once daily and for mortality and morbidity twice daily throughout the study period. The study also tracked body weight (weekly), food consumption (daily), opthalmological status (start and end), hematological and biochemical analyses (start and end), toxicokinetics analysis of plasma (start and end). At the end of the study, all dogs were euthanized by a humane and accepted procedure, subjected to a gross post-mortem examination, organ weights (absolute and relative) were determined, and histopathological examination was carried out.

Results

No symptoms of toxicity observed in the low dose (G2) and control (G1) groups. The toxicokinetic parameters viz. Lambda z, HL Lambda z, T_(max), C_(max), AUC₀₀ of NRC-2694 monohydrochloride on Day 1 was calculated separately for male and female dogs by using WinNonlin version 5.2 software. There was a dose-dependent change in the C_(max) of NRC-2694 monohydrochloride at the three dose levels in both male and female dogs. The median T_(max) for attaining C_(max) at three dose levels (10, 20 and 40 mg/kg) on day 1 were 2, 3, 2 hr in male and 2, 4, 4 hr in female, respectively. The median T_(max) for attaining C_(max) at two dose levels (10 and 20 mg/kg) on day 30 were 2 and 3 hr in male and 2 and 4 hours in female, respectively.

Conclusion

Based on the results of this study, it was concluded that the no observed adverse effect level (NOAEL) of NRC-2694 monohydrochloride in beagle dogs dosed over a period of 30 days is 10 mg/kg b.wt/day when administered orally by capsule. The corresponding value for Erlotinib HCl is 5 mg/kg b.wt/day. Thus, NRC-2694 is advantageously significantly less toxic than Erlotinib HCl in beagle dogs.

Example 12 NRC-2694 is Not Mutagenic in the Ames Test

This study determined that NRC-2694 (the compound of formula IA) is not mutagenic in the Ames test.

Materials and Methods

The present study was conducted by the direct plate incorporation method with five tester strains of Salmonella typhimurium (TA 98, TA 100, TA 102, TA 1535 and TA 1537). NRC-2694 was tested at the doses of 1.2500, 0.3955, 0.1251, 0.0396 and 0.0125 mg/plate. Simultaneously, negative control cultures received DMSO and the respective positive controls received the mutagens 2-Amino anthracene, 2-Nitrofluorene, Sodium azide, 9-Aminoacridine and Mitomycin C (Sigma, St. Louis). In order to study the role of metabolic activation, cultures were incubated both with and without S9 mixture. The induction of Histidine positive colonies was computed and results were statistically treated for comparison.

Results and Conclusion

The study indicated lack of statistically significant induction of His revertant colonies using NRC-2694 in any of the tester strains either with or without S9 addition in the culture when compared to positive and negative controls. Based on the above results, it was concluded that the NRC-2694 monohydrochloride was non-mutagenic according to the Ames bacterial reverse mutation assay.

Example 13 NRC-2694 Lacks Neurotoxicity in the Functional Observational Battery

This study determined that NRC-2694 (the compound of formula IA) showed no evidence of neurotoxicity toward the central and peripheral nervous system in Wistar rats using the functional observations battery (FOB) test.

Materials and Methods

The study was conducted in accordance with “S7A Safety Pharmacology Studies for Human Pharmaceuticals, ICH” July 2001, and as per this study protocol. The FOB assessed the effect of NRC-2694 monohydrochloride on the central and peripheral nervous systems in Wistar rats. Rats that had been fasted for 16 to 18 hours (access to water was not interrupted) were dosed with the compound or water. The rats were administered the compound in an aqueous formulation at three levels, 75, 125 and 250 mg/kg b.wt, as a single oral dose by gavage. The control group received water. The rats were subjected to the FOB 60-120 min. after drug administration.

Animals were subjected to the FOB one at a time except for the multiple activity cage where four animals were placed in four different cages simultaneously. Once complete, the animals were returned to their home cage and the cage placed back on the stand.

Results and Discussion

The functional observational battery tests carried out did not show any adverse effects at the tested doses. NRC-2694A was well tolerated at the given doses having no influence on any of the parameters of the FOB 60-120 minutes post treatment.

The FOB can be employed to assess multiple neurobiological domains including neuromuscular (weakness, in coordination, gait and tremor), sensory (audition, vision and somatosensory), and autonomic (pupil response and salivation) functions. The measures of a neuro behavioral screening battery have been divided into specific functional domains. For example, Lacrimation, salivation, pupil response, palpebral closure, defecation and urination are measures of some aspects of the autonomic system. Similarly the neuro muscular domain can be assessed based on the gait/mobility score, landing foot splay, grip strength and righting reflex; sensorimotor domain based on response to tail pinch and click/touch/approach response; CNS excitability domain based on ease of removal and handling of the animal, clonic/tonic movements, arousal and vocalization; CNS activity domain based on home cage posture, palpebral closure, rearing and motor activity and finally the physiological domain is assessed based on the body weight, body temperature and piloerection.

Example 14 NRC-2694 Lacks Toxicity Against Respiratory Function

This study determined that NRC-2694 (the compound of formula IA) lacks toxicity against respiratory function in Wistar rats.

Materials and Methods

This study evaluated the effect of NRC-2694A monohydrochloride on respiratory function in conscious Wistar rats following administration of a single dose using a whole body plethysmograph. The study was conducted in accordance with “S7A Safety Pharmacology Studies for Human Pharmaceuticals, ICH” issued July 2001. The study used a parallel design with one vehicle and three test item treated groups. NRC-2694 monohydrochloride was administered orally at the doses of 75, 125 and 250 mg/kg body weight. Male Wistar rats were placed in plethysmography chambers and baseline respiratory parameters were recorded for 30 minutes. Following drug administration respiratory parameters were recorded continuously for 3 hours.

Results

At the tested doses NRC-2694 monohydrochloride did not influence any of the observed parameters: respiratory rate, tidal volume, minute volume, and Penh.

Example 15 NRC-2694 Lacks Toxicity Against Cardiovascular Function

This study determined that NRC-2694 (the compound of formula IA) lacks toxicity against cardiovascular function in beagle dogs.

Materials and Methods

This study evaluated the effect of orally administered NRC-2694 monohydrochloride on cardiovascular parameters in telemetered male Beagle dogs. The study was conducted in accordance with ICH “S7A Safety Pharmacology Studies for Human Pharmaceuticals, Note for Guidance on Safety Pharmacology Studies for Human Pharmaceuticals, July 2001”. The doses tested were 0, 15, 30 and 60 mg/kg b.wt. given orally by gelatin capsule. Four male Beagle dogs, each implanted with a TL1IM2-D70-PCT transmitter (Data Sciences International, USA), were used for the study. During the study, the dogs were administered 4 oral treatments representing placebo and all test substance treatment groups with a three day (˜72 hour) wash-out period between each administration, using a Latin square cross-over design.

On dosing days, the telemetered dogs were observed once before commencement of data collection, continuously through 2 hours post-dose remotely and ˜8 hours post-dose for changes in behaviour. Cardiovascular parameters (systolic, diastolic and mean blood pressure, pulse pressure and heart rate) were recorded continuously for ˜60 minutes before dosing and up to 24 hours after dosing. Systolic, diastolic and mean blood pressures, pulse pressure and heart rate were obtained from the femoral artery waveform. Electrocardiograms were recorded continuously for ˜60 minutes before dosing and up to 24 hours after dosing. Electrocardiograms were obtained from subcutaneously placed bio-potential leads in a Lead II configuration. Heart rate and QT intervals were determined from the ECG's.

Results

When administered orally to male Beagle dogs, NRC-2694 monohydrochloride had no significant negative effects on cardiovascular parameters (systolic, diastolic and mean blood pressure, pulse pressure, heart rate, or QT and corrected QT (QTcf) intervals) at the doses tested. The no observed effect level was 60 mg/kg b.wt.

Example 16 NRC-2694 is Safe in a Test of Cardiac Risk Assessment

This study determined that NRC-2694 (the compound of formula IA) is safe in a recombinant cell system used as an index of QT interval prolongation for cardiac risk assessment.

Materials and Methods

The effect of NRC-2694 monohydrochloride on the potassium selective Ikr (tail) current (the rapid component of the delayed rectifier current) was investigated using Chinese hamster ovary (CHO) cells stably transfected with hERG (human ether-a go-go related gene) employing the whole cell patch-clamp technique. Blockade of the Ikr potassium current is considered to constitute an index of QT interval prolongation for cardiac risk assessment. The test was conducted according to S7B: The Nonclinical Evaluation of the Potential for Delayed Ventricular Repolarization (QT Interval Prolongation) By Human Pharmaceuticals. ICH May 2005

This CHO cells stably transfected with hERG were from Flyion GmBH (Germany). Stock cultures of hERG-TRex-CHO cells were stored as frozen permanents in liquid nitrogen. The test compound was evaluated at 10 μM in 0.1% DMSO (v/v). Propafenone (Sigma, St. Louis) at 10 μM was the positive control. The vehicle control was 0.1% (v/v) DMSO in EC solution.

The selection medium was F-12 (Ham)+GlutaMAX, Ph 7.2, 10% FBS, Penicillin (100 U/mL), Streptomycin (100 μg/mL), Blasticidin S HCL (30 μg/mL) and Hygromycin B (400 μg/mL). The growth medium was F-12 (Ham)+GlutaMAX, 10% FBS, Penicillin (100 U/mL) and Streptomycin (100 μg/mL). The induction medium was F-12 (Ham)+GlutaMAX, 10% FBS, Penicillin (100 U/mL) and Streptomycin (100 μg/mL) and Doxycycline (3 μg/mL). The cell dissociation solution was 0.05% Trypsin-EDTA. The extracellular solution (and robot medium) was NMDG 145 mM, KCl 5 Mm, MgCl₂(H₂O)₆ 1 Mm, HEPES 10 mM, Glucose 10 mM, at pH 7.4. The intracellular solution was KMeSO₃ 115 mM, MgCl₂(H₂O)₆ 5 mM, HEPES-KOH 10 mM, EGTA 5 mM, K₂ATP 5 mM, at pH 7.2.

Results and Discussion

The test compound was evaluated at 10 μM where it showed ˜42% inhibition of hERG current. This suggests that NRC-2694, in the experimental context of this study, was an inhibitor of the hERG channel. The positive control, propafenone, caused ˜69% inhibition at 10 μM, indicating the sensitivity of the test system. The tail current mainly reflects the open channel state and is the appropriate index for hERG channel evaluation. Inhibition of the hERG channel can lead to delayed repolarization, which can manifest itself as a prolongation of the QT-interval.

It should be noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing “a compound” includes a mixture of two or more compounds. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.

All publications and patent applications in this specification are indicative of the level of ordinary skill in the art to which this invention pertains.

The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

CHART 1.1 In vitro kinase profiling of NRC-2694 Kinase Target NRC-2694 Descriptive Gene Symbol Ambit Gene Symbol Clone ID (Primary) % Ctrl @ 10000 nM AAK1 AAK1 AAK1 38 ABL1 ABL1 ABL1 67 ABL1 (E255K) ABL1 (E255K) ABL1 (E255K) 80 ABL1 (F317I) ABL1 (F317I) ABL1 (F317I) 48 ABL1 (F317L) ABL1 (F317L) ABL1 (F317L) 33 ABL1 (H396P) ABL1 (H396P) ABL1 (H396P) 34 ABL1 (M351T) ABL1 (M351T) ABL1 (M351T) 37 ABL1 (Q252H) ABL1 (Q252H) ABL1 (Q252H) 15 ABL1 (T315I) ABL1 (T315I) ABL1 (T315I) 35 ABL1 (Y253F) ABL1 (Y253F) ABL1 (Y253F) 23 ABL2 ABL2 ABL2 43 ACVR1 ACVR1 ACVR1 61 ACVR1B ACVR1B ACVR1B 63 ACVR2A ACVR2A ACVR2A 92 ACVR2B ACVR2B ACVR2B 63 ACVRL1 ACVRL1 ACVRL1 91 ADCK3 ADCK3 ADCK3 14 ADCK4 ADCK4 ADCK4 14 AKT1 AKT1 AKT1 100 AKT2 AKT2 AKT2 100 AKT3 AKT3 AKT3 66 ALK ALK ALK 10 AMPK-alpha1 AMPK-alpha1 AMPK-alpha1 50 AMPK-alpha2 AMPK-alpha2 AMPK-alpha2 61 ANKK1 ANKK1 ANKK1 73 ARK5 ARK5 ARK5 29 ASK1 ASK1 ASK1 100 AURKA AURKA AURKA 100 AURKB AURKB AURKB 56 AURKC AURKC AURKC 47 AXL AXL AXL 25 BIKE BIKE BIKE 36 BLK BLK BLK 4.4 BMPR1A BMPR1A BMPR1A 100 BMPR1B BMPR1B BMPR1B 78 BMPR2 BMPR2 BMPR2 100 BMX BMX BMX 73 BRAF BRAF BRAF 81 BRAF (V600E) BRAF (V600E) BRAF (V600E) 73 BRK BRK BRK 46 BRSK1 BRSK1 BRSK1 53 BRSK2 BRSK2 BRSK2 33 BTK BTK BTK 77 CAMK1 CAMK1 CAMK1 65 CAMK1D CAMK1D CAMK1D 83 CAMK1G CAMK1G CAMK1G 75 CAMK2A CAMK2A CAMK2A 63 CAMK2B CAMK2B CAMK2B 77 CAMK2D CAMK2D CAMK2D 35 CAMK2G CAMK2G CAMK2G 58 CAMK4 CAMK4 CAMK4 92 CAMKK1 CAMKK1 CAMKK1 71 CAMKK2 CAMKK2 CAMKK2 60 CDC2L1 CDC2L1 CDC2L1 42 CDC2L2 CDC2L2 CDC2L2 37 CDK11 CDK11 CDK11 87 CDK2 CDK2 CDK2 94 CDK3 CDK3 CDK3 91 CDK5 CDK5 CDK5 100 CDK7 CDK7 CDK7 0.55 CDK8 CDK8 CDK8 100 CDK9 CDK9 CDK9 86 CDKL2 CDKL2 CDKL2 67 CHEK1 CHEK1 CHEK1 42 CHEK2 CHEK2 CHEK2 38 CIT CIT CIT 5.0 CLK1 CLK1 CLK1 31 CLK2 CLK2 CLK2 64 CLK3 CLK3 CLK3 45

CHART 1.2 Kinase Target NRC-2694 Descriptive Gene Symbol Ambit Gene Symbol Clone ID (Primary) % Ctrl @ 10000 nM CLK4 CLK4 CLK4 17 CSF1R CSF1R CSF1R 50 CSK CSK CSK 53 CSNK1A1L CSNK1A1L CSNK1A1L 44 CSNK1D CSNK1D CSNK1D 25 CSNK1E CSNK1E CSNK1E 9.6 CSNK1G1 CSNK1G1 CSNK1G1 55 CSNK1G2 CSNK1G2 CSNK1G2 76 CSNK1G3 CSNK1G3 CSNK1G3 61 CSNK2A1 CSNK2A1 CSNK2A1 56 CSNK2A2 CSNK2A2 CSNK2A2 52 DAPK1 DAPK1 DAPK1 26 DAPK2 DAPK2 DAPK2 61 DAPK3 DAPK3 DAPK3 34 DCAMKL1 DCAMKL1 DCAMKL1 92 DCAMKL2 DCAMKL2 DCAMKL2 69 DCAMKL3 DCAMKL3 DCAMKL3 14 DDR1 DDR1 DDR1 39 DDR2 DDR2 DDR2 76 DLK DLK DLK 92 DMPK DMPK DMPK 6.8 DMPK2 DMPK2 DMPK2 61 DRAK1 DRAK1 DRAK1 51 DRAK2 DRAK2 DRAK2 58 DYRK1B DYRK1B DYRK1B 66 EGFR EGFR EGFR 0 EGFR (E746-A750del) EGFR (E746-A750del) EGFR (E746-A750del) 0 EGFR (G719C) EGFR (G719C) EGFR (G719C) 0 EGFR (G719S) EGFR (G719S) EGFR (G719S) 0 EGFR (L747-E749del, A750P) EGFR (L747-E749del, A750P) EGFR (L747-E749del, A750P) 0 EGFR (L747-S752del, P753S) EGFR (L747-S752del, P753S) EGFR (L747-S752del, P753S) 0.1 EGFR (L747-T751del, Sins) EGFR (L747-T751del, Sins) EGFR (L747-T751del, Sins) 0 EGFR (L858R) EGFR (L858R) EGFR (L858R) 0 EGFR (L861Q) EGFR (L861Q) EGFR (L861Q) 0 EGFR (S752-1759del) EGFR (S752-1759del) EGFR (S752-1759del) 0 EPHA1 EPHA1 EPHA1 55 EPHA2 EPHA2 EPHA2 100 EPHA3 EPHA3 EPHA3 56 EPHA4 EPHA4 EPHA4 64 EPHA5 EPHA5 EPHA5 86 EPHA6 EPHA6 EPHA6 25 EPHA7 EPHA7 EPHA7 70 EPHA8 EPHA8 EPHA8 66 EPHB1 EPHB1 EPHB1 72 EPHB2 EPHB2 EPHB2 83 EPHB3 EPHB3 EPHB3 75 EPHB4 EPHB4 EPHB4 39 ERBB2 ERBB2 ERBB2 31 ERBB4 ERBB4 ERBB4 3.3 ERK1 ERK1 ERK1 98 ERK2 ERK2 ERK2 100 ERK3 ERK3 ERK3 10 ERK4 ERK4 ERK4 3.7 ERK5 ERK5 ERK5 90 ERK8 ERK8 ERK8 62 FAK FAK FAK 85 FER FER FER 94 FES FES FES 72 FGFR1 FGFR1 FGFR1 64 FGFR2 FGFR2 FGFR2 100 FGFR3 FGFR3 FGFR3 100 FGFR3 (G697C) FGFR3 (G697C) FGFR3 (G697C) 82 FGFR4 FGFR4 FGFR4 69 FGR FGR FGR 19 FLT1 FLT1 FLT1 64 FLT3 FLT3 FLT3 49 FLT3 (D835H) FLT3 (D835H) FLT3 (D835H) 8.1 FLT3 (D835Y) FLT3 (D835Y) FLT3 (D835Y) 9.4 FLT3 (ITD) FLT3 (ITD) FLT3 (ITD) 0.1

CHART 1.3 Kinase Target NRC-2694 Descriptive Gene Symbol Ambit Gene Symbol Clone ID (Primary) % Ctrl@10000 nM FLT3 (K663Q) FLT3 (K663Q) FLT3 (K663Q) 56 FLT3 (N8411) FLT3 (N8411) FLT3 (N8411) 16 FLT4 FLT4 FLT4 66 FRK FRK FRK 10 FYN FYN FYN 41 GAK GAK GAK 0.95 GCN2 (Kin.Dom.2.S808G) GCN2 (Kin.Dom.2.S808G) GCN2 (Kin.Dom.2.S808G) 12 GSK3A GSK3A GSK3A 75 GSK3B GSK3B GSK3B 100 HCK HCK HCK 17 HIPK1 HIPK1 HIPK1 70 HPK1 HPK1 HPK1 47 IGF1R IGF1R IGF1R 74 IKK-alpha IKK-alpha IKK-alpha 41 IKK-beta IKK-beta IKK-beta 51 IKK-epsilon IKK-epsilon IKK-epsilon 75 INSR INSR INSR 90 INSRR INSRR INSRR 79 IRAK3 IRAK3 IRAK3 25 ITK ITK ITK 82 JAK1 (JH1domain-catalytic) JAK1 (JH1domain-catalytic) JAK1 (JH1domain-catalytic) 15 JAK1 (JH2domain-pseudokinase) JAK1 (JH2domain-pseudokinase) JAK1 (JH2domain-pseudokinase) 81 JAK2 (JH1domain-catalytic) JAK2 (JH1domain-catalytic) JAK2 (JH1domain-catalytic) 38 JAK3 (JH1domain-catalytic) JAK3 (JH1domain-catalytic) JAK3 (JH1domain-catalytic) 72 JNK1 JNK1 JNK1 99 JNK2 JNK2 JNK2 95 JNK3 JNK3 JNK3 77 KIT KIT KIT 26 KIT (D816V) KIT (D816V) KIT (D816V) 17 KIT (V559D) KIT (V559D) KIT (V559D) 17 KIT (V559D, T670I) KIT (V559D, T670I) KIT (V559D, T670I) 28 KIT (V559D, V654A) KIT (V559D, V654A) KIT (V559D, V654A) 47 LATS1 LATS1 LATS1 91 LATS2 LATS2 LATS2 93 LCK LCK LCK 1.5 LIMK1 LIMK1 LIMK1 62 LIMK2 LIMK2 LIMK2 100 LKB1 LKB1 LKB1 69 LOK LOK LOK 0 LTK LTK LTK 3.8 LYN LYN LYN 13 MAP3K3 MAP3K3 MAP3K3 5.9 MAP3K4 MAP3K4 MAP3K4 53 MAP4K2 MAP4K2 MAP4K2 48 MAP4K3 MAP4K3 MAP4K3 52 MAP4K4 MAP4K4 MAP4K4 20 MAP4K5 MAP4K5 MAP4K5 23 MAPKAPK2 MAPKAPK2 MAPKAPK2 90 MAPKAPK5 MAPKAPK5 MAPKAPK5 79 MARK1 MARK1 MARK1 56 MARK2 MARK2 MARK2 70 MARK3 MARK3 MARK3 62 MARK4 MARK4 MARK4 100 MEK1 MEK1 MEK1 50 MEK2 MEK2 MEK2 44 MEK3 MEK3 MEK3 100 MEK4 MEK4 MEK4 92 MEK6 MEK6 MEK6 75 MELK MELK MELK 57 MERTK MERTK MERTK 48 MET MET MET 33 MINK MINK MINK 16 MKNK1 MKNK1 MKNK1 1.2 MKNK2 MKNK2 MKNK2 16 MLCK MLCK MLCK 77 MLK1 MLK1 MLK1 100 MLK2 MLK2 MLK2 83 MLK3 MLK3 MLK3 72 MRCKA MRCKA MRCKA 81

CHART 1.4 Kinase Target NRC-2694 Descriptive Gene Symbol Ambit Gene Symbol Clone ID (Primary) % Ctrl @ 10000 nM MRCKB MRCKB MRCKB 71 MST1 MST1 MST1 100 MST1R MST1R MST1R 94 MST2 MST2 MST2 85 MST3 MST3 MST3 42 MST4 MST4 MST4 29 MUSK MUSK MUSK 100 MYLK MYLK MYLK 62 MYLK2 MYLK2 MYLK2 4.8 MYO3A MYO3A MYO3A 70 MYO3B MYO3B MYO3B 100 NDR2 NDR2 NDR2 74 NEK1 NEK1 NEK1 66 NEK2 NEK2 NEK2 63 NEK5 NEK5 NEK5 94 NEK6 NEK6 NEK6 94 NEK7 NEK7 NEK7 78 NEK9 NEK9 NEK9 85 NLK NLK NLK 55 p38-alpha p38-alpha p38-alpha 89 p38-beta p38-beta p38-beta 100 p38-delta p38-delta p38-delta 100 p38-gamma p38-gamma p38-gamma 97 PAK1 PAK1 PAK1 74 PAK2 PAK2 PAK2 67 PAK3 PAK3 PAK3 55 PAK4 PAK4 PAK4 80 PAK6 PAK6 PAK6 70 PAK7 PAK7 PAK7 63 PCTK1 PCTK1 PCTK1 100 PCTK2 PCTK2 PCTK2 72 PCTK3 PCTK3 PCTK3 82 PDGFRA PDGFRA PDGFRA 50 PDGFRB PDGFRB PDGFRB 10 PDPK1 PDPK1 PDPK1 94 PFTAIRE2 PFTAIRE2 PFTAIRE2 71 PFTK1 PFTK1 PFTK1 71 PHKG1 PHKG1 PHKG1 50 PHKG2 PHKG2 PHKG2 59 PIK3C2B PIK3C2B PIK3C2B 93 PIK3CA PIK3CA PIK3CA 100 PIK3CA (E545K) PIK3CA (E545K) PIK3CA (E545K) 100 PIK3CB PIK3CB PIK3CB 100 PIK3CD PIK3CD PIK3CD 89 PIK3CG PIK3CG PIK3CG 78 PIM1 PIM1 PIM1 39 PIM2 PIM2 PIM2 83 PIM3 PIM3 PIM3 23 PIP5K1A PIP5K1A PIP5K1A 61 PIP5K2B PIP5K2B PIP5K2B 49 PKAC-alpha PKAC-alpha PKAC-alpha 100 PKAC-beta PKAC-beta PKAC-beta 88 PKMYT1 PKMYT1 PKMYT1 74 PKN1 PKN1 PKN1 50 PKN2 PKN2 PKN2 31 PLK1 PLK1 PLK1 81 PLK3 PLK3 PLK3 97 PLK4 PLK4 PLK4 70 PRKCD PRKCD PRKCD 84 PRKCE PRKCE PRKCE 65 PRKCH PRKCH PRKCH 83 PRKCQ PRKCQ PRKCQ 83 PRKD1 PRKD1 PRKD1 9.6 PRKD2 PRKD2 PRKD2 0.4 PRKD3 PRKD3 PRKD3 7.2 PRKG1 PRKG1 PRKG1 75 PRKG2 PRKG2 PRKG2 40 PRKR PRKR PRKR 51 PRKX PRKX PRKX 100

CHART 1.5 Kinase Target NRC-2694 Descriptive Gene Symbol Ambit Gene Symbol Clone ID (Primary) % Ctrl @ 10000 nM PYK2 PYK2 PYK2 71 RAF1 RAF1 RAF1 100 RET RET RET 65 RET (M918T) RET (M918T) RET (M918T) 37 RET (V804L) RET (V804L) RET (V804L) 81 RET (V804M) RET (V804M) RET (V804M) 100 RIOK1 RIOK1 RIOK1 36 RIOK2 RIOK2 RIOK2 43 RIOK3 RIOK3 RIOK3 37 RIPK1 RIPK1 RIPK1 100 RIPK2 RIPK2 RIPK2 2.4 RIPK4 RIPK4 RIPK4 96 ROCK2 ROCK2 ROCK2 38 ROS1 ROS1 ROS1 46 RPS6KA1 (Kin.Dom.-1-N-terminal) RPS6KA1 (Kin.Dom.-1-N-terminal) RPS6KA1 (Kin.Dom.-1-N-terminal) 100 RPS6KA1 (Kin.Dom.-2-C-terminal) RPS6KA1 (Kin.Dom.-2-C-terminal) RPS6KA1 (Kin.Dom.-2-C-terminal) 49 RPS6KA2 (Kin.Dom.-1-N-terminal) RPS6KA2 (Kin.Dom.-1-N-terminal) RPS6KA2 (Kin.Dom.-1-N-terminal) 70 RPS6KA2 (Kin.Dom.-2-C-terminal) RPS6KA2 (Kin.Dom.-2-C-terminal) RPS6KA2 (Kin.Dom.-2-C-terminal) 78 RPS6KA3 (Kin.Dom.-1-N-terminal) RPS6KA3 (Kin.Dom.-1-N-terminal) RPS6KA3 (Kin.Dom.-1-N-terminal) 94 RPS6KA4 (Kin.Dom.-1-N-terminal) RPS6KA4 (Kin.Dom.-1-N-terminal) RPS6KA4 (Kin.Dom.-1-N-terminal) 84 RPS6KA4 (Kin.Dom.-2-C-terminal) RPS6KA4 (Kin.Dom.-2-C-terminal) RPS6KA4 (Kin.Dom.-2-C-terminal) 8.9 RPS6KA5 (Kin.Dom.-1-N-terminal) RPS6KA5 (Kin.Dom.-1-N-terminal) RPS6KA5 (Kin.Dom.-1-N-terminal) 85 RPS6KA5 (Kin.Dom.-2-C-terminal) RPS6KA5 (Kin.Dom.-2-C-terminal) RPS6KA5 (Kin.Dom.-2-C-terminal) 57 RPS6KA6 (Kin.Dom.-1-N-terminal) RPS6KA6 (Kin.Dom.-1-N-terminal) RPS6KA6 (Kin.Dom.-1-N-terminal) 94 RPS6KA6 (Kin.Dom.-2-C-terminal) RPS6KA6 (Kin.Dom.-2-C-terminal) RPS6KA6 (Kin.Dom.-2-C-terminal) 55 SgK085 SgK085 SgK085 84 SgK110 SgK110 SgK110 83 SIK SIK SIK 53 SIK2 SIK2 SIK2 21 SLK SLK SLK 0.3 SNARK SNARK SNARK 19 SRC SRC SRC 24 SRMS SRMS SRMS 100 SRPK1 SRPK1 SRPK1 40 SRPK2 SRPK2 SRPK2 73 SRPK3 SRPK3 SRPK3 38 STK16 STK16 STK16 100 STK33 STK33 STK33 43 STK35 STK35 STK35 52 STK36 STK36 STK36 4.6 SYK SYK SYK 56 TAK1 TAK1 TAK1 66 TAOK1 TAOK1 TAOK1 54 TAOK3 TAOK3 TAOK3 76 TEC TEC TEC 90 TESK1 TESK1 TESK1 83 TGFBR1 TGFBR1 TGFBR1 83 TGFBR2 TGFBR2 TGFBR2 86 TIE1 TIE1 TIE1 52 TIE2 TIE2 TIE2 76 TLK1 TLK1 TLK1 77 TLK2 TLK2 TLK2 82 TNIK TNIK TNIK 17 TNK1 TNK1 TNK1 41 TNK2 TNK2 TNK2 37 TNNI3K TNNI3K TNNI3K 7.6 TRKA TRKA TRKA 100 TRKB TRKB TRKB 73 TRKC TRKC TRKC 80 TSSK1B TSSK1B TSSK1B 93 TTK TTK TTK 48 TXK TXK TXK 39 TYK2 (JH1domain-catalytic) TYK2 (JH1domain-catalytic) TYK2 (JH1domain-catalytic) 97 TYK2 (JH2domain-pseudokinase) TYK2 (JH2domain-pseudokinase) TYK2 (JH2domain-pseudokinase) 23 TYRO3 TYRO3 TYRO3 42 ULK1 ULK1 ULK1 100 ULK2 ULK2 ULK2 39 ULK3 ULK3 ULK3 62 VEGFR2 VEGFR2 VEGFR2 95

CHART 1.6 Kinase Target Descriptive Clone ID NRC-2694 Gene Symbol Ambit Gene Symbol (Primary) % Ctrl @ 10000 nM WEE1 WEE1 WEE1 74 WEE2 WEE2 WEE2 53 YANK2 YANK2 YANK2 68 YANK3 YANK3 YANK3 100 YES YES YES 41 YSK1 YSK1 YSK1 69 ZAK ZAK ZAK 74 ZAP70 ZAP70 ZAP70 100 

1. A method of inhibiting the growth of a tumor cell in a subject in need thereof, the tumor cell being a pancreatic cancer cell, a HER-2 positive breast cancer cell, or a drug resistant non-small cell lung cancer cell; the method comprising: administering to the subject an effective amount of a compound of formula (I)

in which: R¹ is:

R² is —CH₃ or —CH₂CH₃; or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1, wherein R¹ is:

R² is —CH₃.
 3. The method of claim 1 or 2, wherein the pharmaceutically acceptable salt is a monohydrochloride, a dihydrochloride, or a mixture thereof.
 4. The method of claim 1, 2, or 3, wherein the tumor cell is a drug resistant non-small cell lung cancer cell; and the drug is erlotinib, gefitinib, or a plurality thereof.
 5. The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for treating a subject suffering from growth of a tumor cell, the tumor cell being a pancreatic cancer cell, a HER-2 positive breast cancer cell, or a drug resistant non-small cell lung cancer cell; the use comprising administering to the subject an effective amount of a compound of formula (I):

in which: R¹ is:

R² is —CH₃ or —CH₂CH₃.
 6. The use of claim 5, wherein R¹ is:

R² is —CH₃.
 7. The use of claim 5 or 6, wherein the pharmaceutically acceptable salt is a monohydrochloride, a dihydrochloride, or a mixture thereof.
 8. The use of claim 5, 6, or 7, wherein the tumor cell is a drug resistant non-small cell lung cancer cell; and the drug is erlotinib, gefitinib, or a plurality thereof.
 9. The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a subject suffering from the growth of a tumor cell, the tumor cell being a pancreatic cancer cell, a HER-2 positive breast cancer cell, or a drug resistant non-small cell lung cancer cell;

in which: R¹ is:

R² is —CH₃ or —CH₂CH₃.
 10. The use of claim 9, wherein R¹ is:

R² is —CH₃.
 11. The use of claim 9 or 10, wherein the pharmaceutically acceptable salt is a monohydrochloride, a dihydrochloride, or a mixture thereof.
 12. The use of claim 9, 10, or 11, wherein the tumor cell is a drug resistant non-small cell lung cancer cell; and the drug is erlotinib, gefitinib, or a plurality thereof.
 13. A method of treating drug resistant non-small cell lung cancer in a subject in need of treatment for drug resistant non-small cell lung cancer, the method comprising: administering to the subject an effective amount of a compound of formula (I)

in which: R¹ is:

R² is —CH₃ or —CH₂CH₃; or a pharmaceutically acceptable salt thereof.
 14. The method of claim 13, wherein R¹ is:

R² is —CH₃.
 15. The method of claim 13 or 14, wherein the pharmaceutically acceptable salt is a monohydrochloride, a dihydrochloride, or a mixture thereof.
 16. The method of claim 13, 14, or 15, wherein the drug is erlotinib, gefitinib, or a plurality thereof.
 17. The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for treating a subject suffering from drug resistant non-small cell lung cancer; the use comprising administering to the subject an effective amount of a compound of formula (I):

in which: R¹ is:

R² is —CH₃ or —CH₂CH₃.
 18. The use of claim 17, wherein R¹ is:

R² is —CH₃.
 19. The use of claim 17 or 18, wherein the pharmaceutically acceptable salt is a monohydrochloride, a dihydrochloride, or a mixture thereof.
 20. The use of claim 17, 18, or 19, wherein the drug is erlotinib, gefitinib, or a plurality thereof.
 21. The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a subject suffering from drug resistant non-small cell lung cancer;

in which: R¹ is:

R² is —CH₃ or —CH₂CH₃.
 22. The use of claim 21, wherein R¹ is:

R² is —CH₃.
 23. The use of claim 21 or 22, wherein the pharmaceutically acceptable salt is a monohydrochloride, a dihydrochloride, or a mixture thereof.
 24. The use of claim 21, 22, or 23, wherein the drug is erlotinib, gefitinib, or a plurality thereof.
 25. A method of treating pancreatic cancer in a subject in need of treatment for pancreatic cancer, the method comprising: administering to the subject an effective amount of a compound of formula (I)

in which: R¹ is:

R² is —CH₃ or —CH₂CH₃; or a pharmaceutically acceptable salt thereof.
 26. The method of claim 25, wherein R¹ is:

R² is —CH₃.
 27. The method of claim 25 or 26, wherein the pharmaceutically acceptable salt is a monohydrochloride, a dihydrochloride, or a mixture thereof.
 28. The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for treating a subject suffering from pancreatic cancer; the use comprising administering to the subject an effective amount of a compound of formula (I):

in which: R¹ is:

R² is —CH₃ or —CH₂CH₃.
 29. The use of claim 28, wherein R¹ is:

R² is —CH₃.
 30. The use of claim 28 or 29, wherein the pharmaceutically acceptable salt is a monohydrochloride, a dihydrochloride, or a mixture thereof.
 31. The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a subject suffering from pancreatic cancer;

in which: R¹ is:

R² is —CH₃ or —CH₂CH₃.
 32. The use of claim 31, wherein R¹ is:

R² is —CH₃.
 33. The use of claim 31 or 32, wherein the pharmaceutically acceptable salt is a monohydrochloride, a dihydrochloride, or a mixture thereof.
 34. A method of treating HER-2 positive breast cancer in a subject in need of treatment for HER-2 positive breast cancer, the method comprising: administering to the subject an effective amount of a compound of formula (I)

in which: R¹ is:

R² is —CH₃ or —CH₂CH₃; or a pharmaceutically acceptable salt thereof.
 35. The method of claim 34, wherein R¹ is:

R² is —CH₃.
 36. The method of claim 34 or 35, wherein the pharmaceutically acceptable salt is a monohydrochloride, a dihydrochloride, or a mixture thereof.
 37. The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for treating a subject suffering from HER-2 positive breast cancer; the use comprising administering to the subject an effective amount of a compound of formula (I):

in which: R¹ is:

R² is —CH₃ or —CH₂CH₃.
 38. The use of claim 37, wherein R¹ is:

R² is —CH₃.
 39. The use of claim 37 or 38, wherein the pharmaceutically acceptable salt is a monohydrochloride, a dihydrochloride, or a mixture thereof.
 40. The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a subject suffering from HER-2 positive breast cancer;

in which: R¹ is:

R² is —CH₃ or —CH₂CH₃.
 41. The use of claim 40, wherein R¹ is:

R² is —CH₃.
 42. The use of claim 40 or 41, wherein the pharmaceutically acceptable salt is a monohydrochloride, a dihydrochloride, or a mixture thereof.
 43. A method of inhibiting the growth of a tumor cell in a subject in need thereof, the tumor cell being a pancreatic cancer cell, a HER-2 positive breast cancer cell, or a drug resistant non-small cell lung cancer cell; the method comprising: administering to the subject an effective amount of a compound of formula (IA)

or a pharmaceutically acceptable salt thereof.
 44. The method of claim 43, wherein the pharmaceutically acceptable salt is a monohydrochloride, a dihydrochloride, or a mixture thereof.
 45. The method of claim 43 or 44, wherein the tumor cell is a drug resistant non-small cell lung cancer cell; and the drug is erlotinib, gefitinib, or a plurality thereof.
 46. The use of a compound of formula (IA) or a pharmaceutically acceptable salt thereof for treating a subject suffering from growth of a tumor cell, the tumor cell being a pancreatic cancer cell, a HER-2 positive breast cancer cell, or a drug resistant non-small cell lung cancer cell; the use comprising administering to the subject an effective amount of a compound of formula (IA)


47. The use of claim 46, wherein the pharmaceutically acceptable salt is a monohydrochloride, a dihydrochloride, or a mixture thereof.
 48. The use of claim 46 or 47, wherein the tumor cell is a drug resistant non-small cell lung cancer cell; and the drug is erlotinib, gefitinib, or a plurality thereof.
 49. The use of a compound of formula (IA) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a subject suffering from the growth of a tumor cell, the tumor cell being a pancreatic cancer cell, a HER-2 positive breast cancer cell, or a drug resistant non-small cell lung cancer cell;


50. The use of claim 49, wherein the pharmaceutically acceptable salt is a monohydrochloride, a dihydrochloride, or a mixture thereof.
 51. The use of claim 49 or 50, wherein the tumor cell is a drug resistant non-small cell lung cancer cell; and the drug is erlotinib, gefitinib, or a plurality thereof.
 52. A method of treating drug resistant non-small cell lung cancer in a subject in need of treatment for drug resistant non-small cell lung cancer, the method comprising: administering to the subject an effective amount of a compound of formula (IA)

or a pharmaceutically acceptable salt thereof.
 53. The method of claim 52, wherein the pharmaceutically acceptable salt is a monohydrochloride, a dihydrochloride, or a mixture thereof.
 54. The method of claim 52 or 53, wherein the drug is erlotinib, gefitinib, or a plurality thereof.
 55. The use of a compound of formula (IA) or a pharmaceutically acceptable salt thereof for treating a subject suffering from drug resistant non-small cell lung cancer; the use comprising administering to the subject an effective amount of a compound of formula (IA)


56. The use of claim 55, wherein the pharmaceutically acceptable salt is a monohydrochloride, a dihydrochloride, or a mixture thereof.
 57. The use of claim 55 or 56, wherein the drug is erlotinib, gefitinib, or a plurality thereof.
 58. The use of a compound of formula (IA) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a subject suffering from drug resistant non-small cell lung cancer;


59. The use of claim 58, wherein the pharmaceutically acceptable salt is a monohydrochloride, a dihydrochloride, or a mixture thereof.
 60. The use of claim 58 or 59, wherein the drug is erlotinib, gefitinib, or a plurality thereof.
 61. A method of treating pancreatic cancer in a subject in need of treatment for pancreatic cancer, the method comprising: administering to the subject an effective amount of a compound of formula (IA)

or a pharmaceutically acceptable salt thereof.
 62. The method of claim 61, wherein the pharmaceutically acceptable salt is a monohydrochloride, a dihydrochloride, or a mixture thereof.
 63. The use of a compound of formula (IA) or a pharmaceutically acceptable salt thereof for treating a subject suffering from pancreatic cancer; the use comprising administering to the subject an effective amount of a compound of formula (IA)


64. The use of claim 63, wherein the pharmaceutically acceptable salt is a monohydrochloride, a dihydrochloride, or a mixture thereof.
 65. The use of a compound of formula (IA) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a subject suffering from pancreatic cancer;


66. The use of claim 65, wherein the pharmaceutically acceptable salt is a monohydrochloride, a dihydrochloride, or a mixture thereof.
 67. A method of treating HER-2 positive breast cancer in a subject in need of treatment for HER-2 positive breast cancer, the method comprising: administering to the subject an effective amount of a compound of formula (IA)

or a pharmaceutically acceptable salt thereof.
 68. The method of claim 67, wherein the pharmaceutically acceptable salt is a monohydrochloride, a dihydrochloride, or a mixture thereof.
 69. The use of a compound of formula (IA) or a pharmaceutically acceptable salt thereof for treating a subject suffering from HER-2 positive breast cancer; the use comprising administering to the subject an effective amount of a compound of formula (IA)


70. The use of claim 69, wherein the pharmaceutically acceptable salt is a monohydrochloride, a dihydrochloride, or a mixture thereof.
 71. The use of a compound of formula (IA) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating a subject suffering from HER-2 positive breast cancer;


72. The use of claim 71, wherein the pharmaceutically acceptable salt is a monohydrochloride, a dihydrochloride, or a mixture thereof.
 73. A method of evaluating in vitro kinase activity of quinazoline derivatives of formula (I) essentially as described in the example 6 above.
 74. A method of evaluating in vivo activity of quinazoline derivatives of formula (I) as claimed in claim-1 essentially as described in the examples 7-9 above.
 75. A pharmaceutical composition comprising of a quinazoline derivative of the formula (I), or a pharmaceutically acceptable salt thereof, useful for anticancer treatment as cited in examples 7-9 above. 